Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
The present invention relates generally to the field of NF-xcexaB-dependent activation to regulate immune, inflammatory, and anti-apoptotic responses. In particular, the present invention describes an inhibitor of serine/threonine kinase signaling that regulates NF-xcexaB-dependent activation, thereby regulating immune, inflammatory, and anti-apoptotic responses. The present invention describes several mutant proteins of the NF-xcexaB-inducing kinase (NIK) that prevent activation of a downstream kinase, IxcexaB-specific kinase (IKK). One embodiment of the present invention describes kinase-deficient mutant NIK proteins that inhibits phosphorylation and activation of IKK. Another embodiment of the present invention describes an C-terminal NIK peptide protein that effectively interacts with IKK, and blocks IKK activation by inhibiting NIK/IKK association. Yet another embodiment of the present invention describes a method for inhibiting NF-xcexaB-dependent immune and inflammatory responses using the NIK mutant proteins.
TNFxcex1 (tumor necrosis factor alpha) binding at the plasma membrane induces intracellular signaling events that translate into the induction of NF-xcexaB in the nucleus. The eukaryotic NF-xcexaB/Rel (nuclear factor-xcexaB/Rel) family of transcription factors plays an essential role in the regulation of inflammatory, immune, and apoptotic responses (Bacuerle and Baltimore 1996; Baldwin 1996; Verma et al 1995). One of the distinguishing characteristics of the NF-xcexaB/Rel transcription factor is its posttranslational regulation through interactions with cytoplasmic inhibitory proteins termed IxcexaB (inhibitor-xcexaB). NF-xcexaB corresponds to an inducible eukaryotic transcription factor complex that is negatively regulated in resting cells, by its physical assembly with a family of cytoplasmic ankyrin-rich IxcexaB inhibitors (Bacuerle and Baltimore 1996; Baldwin 1996; Verma et al 1995). Stimulation of cells with various pro-inflammatory cytokines, including TNFxcex1, induces nuclear NF-xcexaB expression. The TNFxcex1-signaling pathway is complex and involves recruitment of at least three adapter proteins, TRADD (TNF-R1 associated death domain protein) and TRAF-2 (TNF-receptor-associated factor)-2, and the serine/threonine kinase RIP (receptor interacting protein) to the cytoplasmic tail of the type 1 TNF receptor (Hsu et al Immunity 1996; Hsu et al Cell 1996) (FIG. 1). In turn, the recruitment of these factors promotes activation of the downstream NIK (Malinin et al 1997) and IKKxcex1 and IKKxcex2 (IxcexaB-specific kinases) (DiDonato et al 1997; Mercurio et al 1997; Regnier et al 1997; Woronicz et al 1997; Zandi et al 1997). The activated IKKxcex1 and IKKxcex2 directly phosphorylates the two N-terminal regulatory serines within IxcexaBxcex1, triggering ubiquitination and rapid degradation of this inhibitor in the 26S proteasome (Bacuerle and Baltimore 1996; Baldwin 1996; Verma et al 1995). Degradation of IxcexaBxcex1 unmasks the nuclear localization signal on NF-xcexaB, allowing the NF-xcexaB to translocate to the nucleus where it engages cognate xcexaB enhancer elements and activates the transcription of various xcexaB-dependent genes involved in inflammatory, immune, and anti-apoptotic responses.
IL-1, a second proinflammatory cytokine, acts in a manner similar to TNFxcex1. IL-1 binding to its receptor recruits the MyD88 and TRAF-6 adapter proteins and (IRAK), a serine-threonine kinase (Cao et al 1996; Muzio et al 1997). Like TRAF-2, TRAF-6 interacts with NIK. Thus, the TNFxcex1 and IL-1 signaling pathways converge at the level of NIK. The present invention provides new insights into the molecular basis for NIK regulation of NF-xcexaB-dependent gene expression, and provides novel methods for modulating NF-xcexaB-dependent immune, inflammatory, and anti-apoptotic responses.
The present invention provides a molecular mechanism to interfere with cytokine induction of NF-xcexaB-dependent immune, inflammatory, and anti apoptotic responses, through interference with the assembly of NIK and the IKK proteins. A relatively small region of NIK selectively impairs the NIK-IKK interaction. The present invention provides a novel and highly specific method for modulating NF-xcexaB-dependent immune and inflammatory responses, based on interruption of the critical proteinxe2x80x94protein interaction of NIK and IKK. The present invention provides methods for inhibiting NF-xcexaB-dependent gene expression, using mutant NIK proteins. One embodiment of the present invention provides kinase-deficient NIK mutant proteins that inhibit activation of IKK. Another embodiment of the invention provides N-terminus NIK mutant proteins that bind IKK, thus inhibiting NIK/IKK interaction.